Agrobacterium tumefaciens-mediated transformation of a hevein-like gene into asparagus leads to stem wilt resistance

Abstract

Asparagus stem wilt, is a significant and devastating disease, typically leading to extensive economic losses in the asparagus industry. To obtain transgenic plants resistant to stem wilt, the hevein-like gene, providing broad spectrum bacterial resistance was inserted into the asparagus genome through Agrobacterium tumefaciens-mediated transformation. The optimal genetic transformation system for asparagus was as follows: pre-culture of embryos for 2 days, inoculation using a bacterial titre of OD600 = 0.6, infection time 10 min and co-culturing for 4 days using an Acetosyringone concentration of 200 μmol/L. Highest transformation frequencies reached 21% and ten transgenic asparagus seedlings carrying the hevein-like gene were identified by polymerase chain reaction. Moreover, integration of the hevein-like gene in the T1 generation of transgenic plants was confirmed by southern blot hybridization. Analysis showed that resistance to stem wilt was enhanced significantly in the transgenic plants, in comparison to non- transgenic plants. The results provide additional data for genetic improvement and are of importance for the development of new disease-resistant asparagus varieties.

Fig 1. The fragments cloned of pBI121-hevein-like vector.

Fig 2. The fragments cloned of the plant expression vector-pCAMBIA3300-35S-hevein-like-NOS.

Fig 3. PCR detection of E.coli colonies carrying the pBI121-hevein-like vector.

M: DNA Marker II; CK: H₂O blank control; H1, H2: positive samples.

Fig 4. Detection of double restriction enzyme digestion for pCAMBIA3300-35S-hevein-like-NOS vector.

M: DNA Marker D2000; CK-: H₂O blank control; CK+: pCAMBIA3300-35S-hevein-like-NOS vector; 1: the size of pCAMBIA3300-35S-hevein-like-NOS vector after double restriction enzyme digestion.

Fig 5. PCR detection for A.tumefaciens transformation.

M: DNA Marker D2000 CK: H₂O blank control 1: Agrobacterium.

Fig 6. Embryo death rate under different PPT concentrations.

Fig 7. Examples of embryo death under different PPT concentrations.

Fig 8. Induction rate of PPT-resistant embryos under different ODs of Agrobacterium suspensions.

Fig 9. Induction rate of PPT-resistant embryos at different Agrobacterium infection times.

Fig 10. Induction rate of PPT-resistant embryos under different co-culture times.

Fig 11. Induction rate of PPT-resistant embryos under different AS concentrations.

Fig 12. Molecular confirmation results of transgenic plants.

(A) Results of PCR detection of transgenic plants M: D2000 DNA Marker CK-: Non-transgenic plants CK+: pCAMBIA3300-35S-hevein-like-NOS vector 1, 2, 3: Transgenic plants (B) Results of southern blot hybridization of T1 generation transgenic plants CK+: pCAMBIA3300-35S-hevein-like-NOS vector CK-: Non-transgenic plants 1, 2, 3: T1 generation transgenic plants.

Fig 13. Process of producing transgenic Asparagus plants.

1: Induction of embryogenic callus; 2: Induction of embryos; 3: A. tumefaciens-mediated genetic transformation of asparagus embryos; 4: Screening of A. tumefaciens-mediated genetic transformation of embryos; 5: Induction of screened transformants; 6: Production of transgenic seedlings; 7: Outdoor transplantation.